Genetic instability plays a prominent role in biology and medicine. It is associated with diverse biological processes such as aging, developmental defects and diseases such as cancer. At the same time, induction of genetic instability with ionizing radiation (IR) is often used to treat cancer. Here, we report that genetic instability has a profound effect on protein quality control by regulating HSP90, one of the two isoforms of HSP90 chaperone protein1. Unlike other chaperones, HSP90 ( and ) acts on only a small fraction of the total proteome called clients . However, because its clients are largely composed of proteins important for signaling, such as kinases, steroid receptors, transcription factors and enzymes, the impact of HSP90 activity extends far beyond the relatively small number of its clients. The two HSP90 isoforms in vertebrates have different expression patterns: HSP90, the major inducible form, is not essential for viability2 but HSP90, the minor constitutive form, is essential for viability. Other than the distinct expression patterns, functional differences between the two HSP90 isoforms are poorly understood. Here, we show that phosphorylation of Thr 5 and 7 in HSP90 by DNA-activated protein kinase (DNA-PK) in response to double-strand DNA-breaks (DSBs) increases the ATPase activity of HSP90 by dimerizing the N-terminal domain. This phosphorylation disrupts many HSP90-client complexes in response to DSBs and facilitates a vigorous response to DSBs, including heat shock response and apoptosis. However, disruption of the HSP90-client complex decreases folding of some clients such as the cystic fibrosis transmembrane conductance regulator (CFTR) and glucocorticoid receptor (GR). A survey of 40 strong HSP90 kinase clients indicates that DNA DSBs disrupt the HSP90 complex for at least 40% of the clients. These findings indicate that DNA-PK and HSP90 mediate an unexpected coordination between protein quality control and genetic integrity.